LINE-1 elements (L1s) are abundant retrotransposons that comprise approximately 20% of mammalian genomes. Active L1 retrotransposons can impact the genome in a variety of ways, creating insertions, deletions, new splice sites or gene expression fine-tuning. The predominant view has been that most evolutionarily significant L1 retrotransposition is restricted germ cells or early embryos. However, our original findings suggest that germ cells may be protected against new L1 insertions and most insertions actually happen in somatic tissues, especially in the brain. Here we show preliminary data indicating that methyl-CpG-binding protein 2 (MeCP2), which is associated with Rett Syndrome (RTT), can function as a negative regulator of L1 expression in neural stem cells, leading to increased neuron-specific genetic variation in rodents and humans. Neurons lacking MeCP2 not only have more insertions per cell but also show misregulation of post-insertion, possibly giving rise to some of the phenotypes seen in individuals with RTT. We hypothesize that L1 integration events in neurons are tightly controlled by MeCP2. Furthermore, in neurons devoid of functional MeCP2, the control of L1 mutagenesis due to de novo integration is lost, contributing to improper neuronal function. To test this hypothesis, we propose two specific aims: 1) To determine the molecular mechanism involved in L1 mobility in neurons. Specifically, we plan on revealing the mechanistic interaction and regulation of MeCP2 on L1 transcription, retrotransposition, and post-integration silencing during neuronal differentiation, and 2) To determine a possible contribution of L1 de novo insertions to neuronal networks. Here, we plan on defining the impact of neuronal L1 retrotransposition by generating tools to prevent L1 expression in NPCs. We will explore these tools to assess the impact of L1 insertions in both normal and MeCP2 knockout (KO) backgrounds. The main goal of the proposal is to define the molecular mechanism behind the specific L1 retrotransposition in neurons and to elucidate the effects of such activity in the brain. Unveiling the consequence of L1 integration events on the mammalian nervous system could help explain how complex behavior and individuality arises from random genetic and molecular processes. Understanding the regulation of L1 expression and de novo integration will yield insights into human neurological disease, such as Rett syndrome. PUBLIC HEALTH RELEVANCE: This proposal describes a series of experiments to test the hypothesis that L1 neuronal retrotransposition can contribute to genetic diversity and affect networks. The outcome of this study will increase our understanding of human cognitive diversity and may contain insights into the genesis of multiple neurological diseases.